Detection and quantification of nucleic acids using PCR can be useful for a large number of biological and clinical applications. The first generation of PCR users performed end-point analysis by gel electrophoresis to obtain qualitative results. The advent of real-time PCR (which can involve monitoring the progression of amplification using fluorescence probes) permitted quantification of nucleic acids in some settings. Quantitative information can be obtained from the cycle threshold (CT), a point on the analogue fluorescence curve where the signal increases above background. Real-time PCR can involve the use of external calibrators or normalization to endogenous controls.
In digital PCR, a sample containing PCR reagents and target nucleic acid molecules can be distributed across multiple replicate partitions, often such that each individual partition contains on average less than one target nucleic acid molecule. After amplification, reactions containing one or more templates can be detectable and can emit a signal such as a fluorescent signal.
In some cases, digital PCR can be accomplished by a technique called droplet digital PCR. The operations for droplet digital PCR can involve (1) partitioning a fluid sample containing PCR reagents and nucleic acid target molecule(s) into multiple droplets, (2) performing an amplification cycle on the droplets, and (3) detecting the presence of nucleic acids in the droplets. A nucleic acid sample can be partitioned into multiple droplets using oil and emulsion chemistry. For example, an aqueous sample can be partitioned into multiple emulsified droplets in a continuous oil phase using microfluidics technologies.